The invention relates to phase fluorimetry or frequency domain fluorimetry.
Frequency Domain fluorimetry is a detection technique that measures the time lag between absorption and emission of photons thereby determining the lifetime of a fluorophore. A fluorophore is defined as a member of an atomic group with one excited molecule that emits photons and is fluorescent. The excitation modulation will result in an emission delay relative to excitation, and can be measured as a phase shift. The lifetime is determined by measuring the phase shift and amplitude of the fluorescence when an excitation source is sinusoidally modulated.
Frequency Domain (FD) Fluorimetry, capitalizes on the frequency response function of a fluorophore and offers independence from light scatter and excitation/emission intensity variations in order to extract a sample's fluorescent lifetime. Light scatter can be a significant problem in pulsed excitation measurements because the intense laser pulse can overwhelm the weak fluorescent signal. This problem is alleviated in continuous wave (CW) frequency domain fluorimetry measurements because the excitation source is not as intense. In addition, since the method ratios the fluorescence to the excitation intensity as a function of modulation frequency, any variations in excitation intensity will be automatically removed from the data. Samples which fluoresce in the visible range are commonly excited with ultraviolet laser sources, which can be problematic because they are not typically high power, portable devices.
The simplest model for the temporal response of a fluorophore is a single exponential decay. By abruptly terminating the excitation source (for example, a pulsed ultraviolet laser) and then observing the fluorescent intensity as a function of time, the decay can be observed, averaged and stored on a digitizing oscilloscope. The fluorescent lifetime, τ, can be measured by fitting the decaying amplitude to an exponential decay:
                              I          ⁡                      (            t            )                          =                              A            0                    ⁢                      exp            ⁡                          (                              -                                  t                  τ                                            )                                                          Eq        ⁢                                  ⁢                  (          1          )                    
An operator must ensure that the excitation source turns off much more quickly than τ and that the measurement equipment is much faster than τ. Of course the lifetime of the fluorophore will depend on the properties of the host medium due to nonradiative relaxation mechanisms. The single exponential decay modeled above is actually an ensemble average of many fluorophores in the sample (i.e., there is actually a range of lifetimes observed in the sample, but we will assume that these can be averaged into a single decay lifetime). It is assumed in this research effort that the multi-exponential decay times are separated by less than 20 percent such that a single exponential decay is a valid mean value for the fluorophore.
Frequency Domain fluorimetry is a technique that determines the lifetime of a fluorophore by measuring the phase shift and amplitude of the fluorescence when the excitation source is sinusoidally modulated. The excitation modulation will result in an emission delay relative to excitation and can be measured as a phase shift. A sinusoidal excitation source with modulation frequency, ω, will result in a frequency dependent fluorescence of:I(t)∝m(ω)sin(ωt+φ(ω)),  Eq (2)
where the phase delay, φ(ω), and modulation depth, m(ω), are determined by the lifetime of the fluorophore and the frequency by:
                              tan          ⁡                      (                          ϕ              ⁡                              (                ω                )                                      )                          =                              ω            ⁢                                                  ⁢            τ            ⁢                                                  ⁢            and            ⁢                                                  ⁢                          m              ⁡                              (                ω                )                                              =                                    1                                                (                                      1                    +                                                                  ω                        2                                            ⁢                                              τ                        2                                                                              )                                                      .                                              Eq        ⁢                                  ⁢                  (          3          )                    
In traditional Frequency Domain fluorimetry, the modulation frequency is varied and the phase and modulation amplitude are measured. Alternatively, the in-phase and quadrature components can be recorded and the lifetime calculation is extracted by least squares analysis of recorded data using the in-phase, Nω, and quadrature, Dω, amplitudes of the fluorescent intensity by:
                                          N            ω                    =                                                                                          ∫                    0                    ∞                                    ⁢                                                            I                      ⁡                                              (                        t                        )                                                              ⁢                                          sin                      ⁡                                              (                                                  ω                          ⁢                                                                                                          ⁢                          t                                                )                                                              ⁢                                                                                  ⁢                                          ⅆ                      t                                                                                                            ∫                    0                    ∞                                    ⁢                                                            I                      ⁡                                              (                        t                        )                                                              ⁢                                          ⅆ                      t                                                                                  ⁢                                                          ⁢              and              ⁢                                                          ⁢                              D                ω                                      =                                                            ∫                  0                  ∞                                ⁢                                                      I                    ⁡                                          (                      t                      )                                                        ⁢                                      cos                    ⁡                                          (                                              ω                        ⁢                                                                                                  ⁢                        t                                            )                                                        ⁢                                      ⅆ                    t                                                                                                ∫                  0                  ∞                                ⁢                                                      I                    ⁡                                          (                      t                      )                                                        ⁢                                      ⅆ                    t                                                                                      ,                            Eq        ⁢                                  ⁢                  (          4          )                    where the experimental values of φc(ω) and mc(ω) are given by
                              tan          ⁡                      (                          ϕ              c                        )                          =                                                            N                ω                                            D                ω                                      ⁢                                                  ⁢            and            ⁢                                                  ⁢                          m              c                                =                                                    (                                                      N                    ω                    2                                    +                                      D                    ω                    2                                                  )                                            1                /                2                                      .                                              Eq        ⁢                                  ⁢                  (          5          )                    
The lifetime can then be calculated such that χ2 is minimized in
                                          χ            2                    =                                                    1                ν                            ⁢                                                ∑                  ω                                ⁢                                                      (                                                                  ϕ                        -                                                  ϕ                          c                                                                    δϕ                                        )                                    2                                                      +                                          1                ν                            ⁢                                                ∑                  ω                                ⁢                                                      (                                                                  m                        -                                                  m                          c                                                                                            δ                        ⁢                                                                                                  ⁢                        m                                                              )                                    2                                                                    ,                            Eq        ⁢                                  ⁢                  (          6          )                    where the values of δφ and δm represent the uncertainty in the measured values, and v is the number of degrees of freedom.
As claimed and disclosed in the present invention, mercury vapor lamps, a common source of industrial facility lighting, emit radiation that overlaps the UV/blue absorption spectrum of many fluorophores and may be used as an efficient and portable excitation source. The AC power modulation of mercury vapor lamps modulates the lamp's intensity at 120 Hz (in the United States) and higher harmonics. The present invention offers a means to remotely detect fluorophores such as uranium from distances exceeding a kilometer. The present invention provides an option to exploiting Frequency Domain fluorimetry remotely and overcomes problems in the art of using high power laser sources by using commonly available equipment, often available at the site in question.